Study of membrane proteins at the molecular level is one of the major challenges for today's biologists. ATP-binding cassette (ABC) transporters are one of the largest and most diverse superfamily of membrane proteins found in all living organisms ranging from bacteria to human. All ABC transporters share a common structure organization, suggesting a similar mechanism of energy coupling. ABC transporters are medically relevant. For instance, they are responsible for severe sicknesses and multidrug resistance in bacteria and cancer chemotherapy. Despite extensive studies, the molecular mechanism of ABC transporters remains elusive and many questions remain unanswered. In this proposal, we aim to develop single nanoparticle optical assays for real-time characterizing the functioning mechanism of BmrA, a multidrug bacterial transporter belonging to the ABC transporter superfamily. The specific research aims are described below: Aim 1: We will develop the optimum nanoparticle probes for tracing the transport mechanisms and kinetics of BrmA (WT) and their mutants in real-time at the single transporter resolution with high temporal and spatial information, aiming to determine individual steps of the catalytic cycle directly responsible for substrate translocation. Combining with high-resolution transmission electron microscopy (TEM) imaging, we aim to depict the transport pathways and verify proposed models. Aim 2: We aim to use the optimum nanoparticle probes developed in Aim 1 to trace the real-time transport mechanism and kinetics of drugs and genes via the ABC transporter. In addition, we will determine the nanoparticles that can escape the extrusion mechanisms of ABC transporter, and use them as carriers to deliver genes and drugs into living cells, aiming to explore the effective delivery means for better therapy. Aim 3: We will study the biocompatibility of nanoparticles at the cellular and genomic level, aiming to design biocompatible nanoparticles for molecular study of the ABC transports in living cells in real-time. The outcomes include: new nanoparticle assays for real-time measurement of membrane transport pathways and mechanisms of membrane transporters;better understanding of fundamental mechanism of ABC membrane transport;and effective means for the delivery of genes and drugs into living cells.